Role of the Rab family small G proteins in cell polarity and adhesion

紧密连接(TJ)是上皮细胞之间的粘附点，确保顶端和基底侧质膜(PM)区域的发育和维持。构成TJs的蛋白包括介导细胞间黏附的跨膜蛋白和连接到细胞骨架并参与细胞内信号转导的胞质斑块蛋白。TJs的跨膜蛋白由occludin和claudins组成。Rab家族小G蛋白由哺乳动物细胞中的60多个家族成员组成，在决定囊泡运输途径的特异性方面起着至关重要的作用。Rab家族的两个成员Rab3B和Rab13定位于TJs，位于极化的上皮细胞和非极化的成纤维细胞的胞浆泡结构中，但其功能尚不清楚。在本研究中，我们研究了Rab3B和Rab13在调节TJ跨膜蛋白囊泡运输中的作用。在胞吐途径中，我们发现Rab3B和Rab13分别引导基侧跨膜蛋白、低密度脂蛋白受体和claudin-1的TH细胞表面转运。我们还发现，在上皮细胞中，封闭素池不断地被内吞并循环回到细胞表面。我们的结果表明，Rabl3，而不是Rab3B，特异性地调节了occludin的内吞循环。生化内吞和循环分析表明，Rab13指导内吞的闭塞蛋白的循环，但不指导其内吞。此外，我们发现Rab13不影响转铁蛋白受体的循环，转铁蛋白受体被循环到底侧PM结构域。我们分离到一个与GTP结合形式的Rab13特异相互作用的蛋白质，但不能与其GDP结合形式相互作用。我们的结果表明，Rab13是阻滞素和Claudins转运到TJ的关键调节因子。

Tight junctions (TJs) are points of adhesion between epithelial cells that ensure the development and maintenance of the apical and basolateral plasma membrane (PM) domains. Proteins constituting TJs include the transmembrane proteins mediating cell-cell adhesion and the cytosolic plaque proteins linking to the cytoskeleton and participating in the intracellular signaling. Occludin and claudins constitute the transmembrane proteins in TJs. The Rab family small G proteins, which consist of more than 60 family members in mammalian cells, play a crucial role in etermining the specificity of vesicular transport pathways. Two Rab family members, Rab3B and Rab13, localize to TJs in polarized epithelial cells and cytoplasmic vesicular structures in non-polarized fibroblasts, but their functions are poorly understood. In the present study, we have examined the role of Rab3B and Rab13 in regulating the vesicular transport of TJ transmembrane proteins. In the exocytotic pathways, we found that Rab3B and Rab13 direct th cell-surface transport of a basolateral transmembrane protein, low-density lipoprotein receptor, and claudin-1, respectively. We also found that a pool of occludin was continuously endocytosed and recycled back to the cell-surface in epithelial cells. Our results indicated that Rabl3, but not Rab3B, specifically regulated the endocytic recycling of occludin. Biochemical endocytosis and recycling assays demonstrated that Rab13 directed the recycling of endocytosed occludin, but not its endocytosis. Furthermore, we found that Rab13 did not affect the recycling of transferrin receptor, that was recycled to the basolateral PM domain. We isolated a protein that was specifically interacted with GTP-bound form of Rab13, but not its GDP-bound form. Our results suggested that Rab13 was a crucial regulator for the transport of occludin and claudins to TJs.